1. Field of the Invention
The present invention relates generally to assays and more specifically to displacement-type assays.
2. Description of the Background Art
U.S. Pat. No. 5,183,740, incorporated in its entirety herein for all purposes, describes a flow immunoassay system and method for performing displacement immunoassays. In a displacement assay, unlike a competitive assay, the antibody is exposed to labelled analyte prior to exposure to analyte. The analyte is in contact with the antibody and labelled, bound analyte an insufficient amount of time to establish equilibrium.
Because no time needs to be dedicated to establishing equilibrium, displacement assays are faster than competitive assays. A displacement assay, however, generally provides a smaller signal than a competitive assay. In a displacement assay, the available binding sites of the antibody are saturated or nearly saturated with labelled analyte before the unlabelled analyte is added. Since equilibrium (with labelled analyte and unlabelled analyte continually binding, releasing and competing with each other for rebinding to the available binding sites on the antibody in a steady state) has not been achieved, most of the labelled analyte in a displacement assay remains bound to the antibody and unable to provide a signal.
The relatively small signal provided by the displacement assay places an additional value on assuring the consistency of assay conditions. The bead-containing columns described in U.S. Pat. No. 5,183,740 for displacement assays must be carefully stored, prepared, and loaded to assure chemical and physical consistency (i.e., porosity, avoidance of channeling) from test to test. The need for this careful preparation and testing increases the labor, skill, and costs needed to perform accurate displacement assays. Additionally, the problems associated with the use of bead-containing columns limit the lower detection limit for displacement assays.
In studies performed at US Drug Testing, Inc. (Rancho Cucamonga, Calif.), better results for a displacement assay were achieved using tall, thin columns of beads coated with an antibody and labelled antigen than with short, wide columns. Furthermore, the efficiency with which the labelled antigen dissociated from antibody in the presence of unlabelled antigen was greater when flow rates were reduced and the antigen had more time to interact with the immobilized complex (Wemhoff et al. J. Immunol. Methods, 223-230, 1992). Both of these sets of experiments suggested that immobilization of the antibody and labelled antigen on a porous membrane would not provide a suitable matrix for the displacement assay since this geometry would not allow sufficient time, under flow conditions, for the antigen to interact efficiently with the complex to displace detectable amounts of the labelled antigen.
U.S. Pat. No. 5,369,007, to David A. Kidwell discloses a displacement assay in which samples pass through a membrane having an antibody immobilized thereon. The binding sites of the immobilized antibody are bound to an enzymatically labelled analyte. Analyte from the sample displaces the labelled analyte, causing the labelled analyte and the remainder of the sample to pass into a superabsorbent layer. The superabsorbent layer contains a substrate for the enzymatic label and any needed indicator. The Kidwell patent, however, teaches the need for a flow rate of about 0.02 ml/min and interaction times of about 1 to 5 min to assure a detectable interaction between the analyte and the antibody. In many situations, even faster results are desirable. Additionally, the Kidwell microassay card is not reusable.
Accordingly, it is an object of this invention to perform bioassays capable of detecting minute quantities of an analyte in under one minute.
It is another object of the present invention to quickly perform bioassays in a format that allows reuse of the matrix that selectively binds the analyte.
These and additional objects of the invention are accomplished by quickly flowing a sample past a non-absorbent membrane having a binding element covalently bound thereto to form attachment sites for the analyte. The available attachment sites are essentially saturated with a labelled form of the analyte. Nonspecific binding sites are blocked to prevent nonspecific binding. Additionally, the sample flows past the membrane at a rate greater than that needed to achieve equilibrium between the dissociation of labelled analyte from the binding sites and the attachment of analyte (labelled or unlabelled) thereto. The processed sample is then analyzed for the presence of any labelled antigen that the unlabelled analyte has displaced from its binding site. This analysis can be qualitative or quantitative.